Method enabling use of extracellular RNA extracted from plasma or serum to detect, monitor or evaluate cancer

ABSTRACT

This invention relates to the use of tumor-derived or associated extracellular ribonucleic acid (RNA) found circulating in the plasma or serum fraction of blood for the detection, monitoring, or evaluation of cancer or premalignant conditions. Extracellular RNA may circulate as non-bound RNA, protein-bound RNA, lipid-RNA complexes, lipoprotein (proteolipid)-RNA complexes, protein-RNA complexes including within or in association with ribonucleoprotein complexes, nucleosomes, or within apoptotic bodies. Any intracellular RNA found in plasma or serum can additionally be detected by this invention. Specifically, this invention enables the extraction of circulating RNA from plasma or serum and utilizes nucleic acid amplification assays for the identification, detection, inference, monitoring, or evaluation of any neoplasm, benign, premalignant, or malignant, in humans or other animals, which might be associated with that RNA. Further, this invention allows the qualitative or quantitative detection of tumor-derived or associated extracellular RNA circulating in the plasma or serum of humans or animals with or without any prior knowledge of the presence of cancer or premalignant tissue.

This application claims priority as a continuation of U.S. Ser. No.11/216,858, filed Aug. 31, 2005, which is a continuation of U.S. Ser.No. 10/013,868, filed Oct. 30, 2001, now U.S. Pat. No. 6,939,671,granted Sep. 6, 2005, which is a continuation of U.S. Ser. No.09/155,152, filed Sep. 22, 1998, now U.S. Pat. No. 6,329,179, grantedDec. 11, 2001, which claims priority to U.S. Ser. No. 60/014,730, filedMar. 26, 1996, the disclosures of each of which are explicitlyincorporated by reference herein.

BACKGROUND OF THE INVENTION

Ribonucleic acid (RNA) is essential to the processes which allowtranslation of the genetic code to form proteins necessary for allcellular functions, both in normal and neoplastic cells. While thegenetic code structurally exists as deoxyribonucleic acid (DNA), it isthe function of RNA, existing as the subtypes transfer-RNA,messenger-RNA or messenger-like RNA, and ribosomal-RNA, to carry andtranslate this code to the cellular sites of protein production. In thenucleus, this RNA may further exist as or in association withribonucleoproteins (RNP). The pathogenesis and regulation of cancer isdependent upon RNA-mediated translation of specific genetic codes, whichoften reflects mutational events within oncogenes, to produce proteinsinvolved with cell proliferation, regulation, and death. Furthermore,other RNA and their translated proteins, although not necessarily thoseinvolved in neoplastic pathogenesis or regulation, may serve todelineate recognizable characteristics of particular neoplasms by eitherbeing elevated or inappropriately expressed. Thus, recognition ofspecific RNA can enable the identification, detection, inference,monitoring, or evaluation of any neoplasm, benign, malignant, orpremalignant, in humans and animals. Furthermore, since RNA can berepetitively created from its DNA template, for a given gene within acell there may be formed a substantially greater number of associatedRNA molecules than DNA molecules. Thus, an RNA-based assay should havegreater sensitivity, and greater clinical utility, than its respectiveDNA-based assay. Note that the term RNA denotes ribonucleic acidincluding fragments of ribonucleic acid consisting of ribonucleic acidsequences.

RNA based nucleic acid amplification assays, including the reversetranscriptase polymerase chain reaction (RT-PCR, also known as reversetranscription polymerase chain reaction or RNA-PCR), branched DNA signalamplification, and self-sustained sequence replication assays, such asisothermal nucleic acid sequence based amplification (NASBA), haveproven to be highly sensitive and specific methods for detecting smallnumbers of RNA molecules. As such, they can be used in direct assays ofneoplastic tissue (Mori et al., 1995, Detection of cancermicrometastases in lymph nodes by reverse transcriptase-polymerase chainreaction. Cancer Res 55:3417-3420; Higashiyama et al., 1995, Reducedmotility related protein-1 (MRP-1/CD9) gene expression as a factor ofpoor prognosis in non-small cell lung cancer. Cancer Res 55:6040-6044;Ozcelik et al., 1995, Low levels of expression of an inhibitor ofcyclin-dependent kinases (CIP1/WAF1) in primary breast carcinomas withp53 mutations. Clin Cancer Res 1:907-912). Since peripheral blood isreadily obtainable from patients with cancer, and metastatic cancercells are known to circulate in the blood of patients with advancedcancer, several investigators have recently used RT-PCR to detectintracellular RNA extracted from circulating cancer cells (Smith et al.,1991, Detection of melanoma cells in peripheral blood by means ofreverse transcriptase and polymerase chain reaction. Lancet338:1227-1229; Datta et al., 1994, Sensitive detection of occult breastcancer by the reverse-transcriptase polymerase chain reaction. J ClinOncol 12:475-482; Moreno et al., 1992, Detection of hematogenousmicrometastasis in patients with prostate cancer. Cancer Res52:6110-6112; Ghossein et al., 1995, Detection of circulating tumorcells in patients with localized and metastatic prostatic carcinoma:clinical implications. J Clin Oncol 13:1195-1200). It must be emphasizedthat currently investigators apply RT-PCR to detect extractedintracellular RNA from a predominately cellular fraction of blood inorder to demonstrate the existence of circulating cancer cells. RT-PCRis applied only to the cellular fraction of blood obtained from cancerpatients, i.e., the cell pellet or cells within whole blood. The plasmaor serum fraction of blood is usually discarded prior to analysis, butis not examined separately since such a cellular fraction approachrelies upon the presence of metastatic circulating cancer cells, it isof limited clinical use in patients with early cancers, and is notuseful in the detection of non-invasive neoplasms or pre-malignantstates.

The invention described by this patent application demonstrates thenovel use of that human or animal tumor-derived or tumor-associated RNAfound circulating in the plasma or serum fraction of blood, as a meansto detect, monitor, or evaluate cancer and premalignant states. Thisinvention is based upon the application of RNA extraction techniques andnucleic acid amplification assays to detect tumor-derived or associatedextracellular RNA found circulating in plasma or serum. In contrast tothe detection of viral-related RNA in plasma or serum, and the detectionof tumor-associated DNA in plasma and serum, the detection of human ormammalian RNA, and particularly tumor-derived or associated RNA, hasnever been detected specifically within the plasma or serum fraction ofblood using nucleic acid amplification methodology, and thus representsa novel and non-obvious use for these RNA extraction methods and nucleicacid amplification assays. Since this invention is not dependent uponthe presence of circulating cancer cells, it is clinically applicable tocases of early cancer, non-invasive cancers, and premalignant states, inaddition to cases of invasive cancer and advanced cancer. Further, thisinvention allows the detection of RNA in previously frozen or otherwisestored plasma and serum, thus making plasma and serum banks availablefor analysis and otherwise increasing general usefulness.

Tumor-derived or tumor-associated RNA that is present in plasma andserum may exist in two forms. The first being extracellular RNA, but thesecond being extractable intracellular RNA from cells occasionallycontaminating the plasma or serum fraction. In practice, it is notnecessary to differentiate between intracellular and extracellular inorder to detect RNA in plasma or serum using the invention, and thisinvention can be used for detection of both. The potential uses oftumor-derived or associated extracellular RNA have not been obvious tothe scientific community, nor has the application of nucleic acidamplification assays to detect tumor-derived or associated extracellularRNA been obvious. Indeed, the very existence of tumor-derived orassociated extracellular RNA has not been obvious to the scientificcommunity, and is generally considered not to exist. It is generallybelieved that plasma ribonucleases rapidly degrade any extracellularmammalian RNA which might circulate in blood, rendering it nondetectable(Reddi et al., 1976, Elevated serum ribonuclease in patients withpancreatic cancer. Proc Nat Acad Sci USA 73:2308-2310). Komeda et al.,for example, specifically added free RNA to whole blood obtained fromnormal volunteers, but were unable to detect that RNA using PCR (Komedaet al., 1995, Sensitive detection of circulating hepatocellularcarcinoma cells in peripheral venous blood. Cancer 75:2214-2219).However, nucleases appear inhibited in the plasma of cancer patients(Leon et al., 1981, A comparison of DNA and DNA-binding protein levelsin malignant disease. Europ J Cancer 17:533-538). In addition,extracellular RNA, either complexed to lipids and proteolipids,protein-bound, or within apoptotic bodies, would be protected fromribonucleases. Thus, although still undefined, tumor-derived orassociated extracellular RNA may be present in plasma or serum viaseveral mechanisms. Extracellular RNA could be secreted or shed fromtumor in the form of lipoprotein (proteo-lipid)-RNA or lipid-RNAcomplexes, it could be found within circulating apoptotic bodies derivedfrom apoptotic tumor cells, it could be found in proteo-RNA complexesreleased from viable or dying cells including or in association withribonucleoproteins, or in association with other proteins such asgalectin-3, or RNA could be released from necrotic cells and thencirculate bound to proteins normally present in plasma. Additionally itcould exist circulating within RNA-DNA complexes including thoseassociated with ribonucleoproteins and other nucleic RNA. Further, RNAmay exist within several of these moieties simultaneously. For example,RNA may be found associated with ribonucleoprotein found withinproteo-lipid apoptotic bodies. The presence of extracellular RNA inplasma or serum makes their detection by nucleic acid amplificationassays feasible.

Several studies in the literature support the existence of tumor-derivedor associated extracellular RNA. RNA has been shown to be present on thecell surface of tumor cells, as demonstrated by electrophoresis (Juckett& Rosenberg, 1982, Actions of cis-diamminedichloroplatinum on cellsurface nucleic acids in cancer cells as determined by cellelectrophoresis techniques. Cancer Res 42:3565-3573), membranepreparations (Davidova & Shapot, 1970, Liporibonucleoprotein complex asan integral part of animal cell plasma membranes. FEBS Lett. 6:349-351),and ³²P release (Rieber & Bacalo, 1974, An “external” RNA removable frommammalian cells by mild proteolysis. Proc Natl Acad Sci USA71:4960-4964). Shedding of phospholipid vesicles from tumor cells is awell described phenomena (Taylor & Blak, 1985, Shedding of plasmamembrane fragments. Neoplastic and developmental importance. In: TheCell Surface in Development and Cancer, Develop Biol Vol 3, pp. 33-57.Editor: M. S. Steinberg. Plenum Press, New York, London; Barz et al.,1985, Characterization of cellular and extracellular plasma membranevesicles from a non-metastasing lymphoma (Eb) and its metastasingvariant (Esb). Biochim. Biophys. Acta 814:77-84), and similar vesicleshave been shown to circulate in the blood of patients with cancer (Carret al., 1985, Circulating membrane vesicles in leukemic blood. CancerRes 45:5944-5951). Kamm and Smith used a fluorometric method toquantitate RNA concentrations in the plasma of healthy individuals (Kamm& Smith, 1972, Nucleic acid concentrations in normal human plasma.Clinical Chemistry 18:519-522). Rosi and colleagues used high resolutionnuclear magnetic resonance (NMR) spectroscopy to demonstrate RNAmolecules complexed with lipid vesicles which were shed from a humancolon adenocarcinoma cell line (Rosi et al., 1988, RNA-lipid complexesreleased from the plasma membrane of human colon carcinoma cells. CancerLett. 39:153-160). Further characterization of these lipid-RNA complexesdemonstrated the vesicles additionally contained triglycerides,cholesterol esters, lipids, oligopeptide, and phospholipids (Masella etal., 1989, Characterization of vesicles, containing an acylatedoligopeptide, released by human colon adenocarcinoma cells. FEBS Lett.246:25-29). Mountford et al. used magnetic resonance spectroscopy toidentify a proteolipid in the plasma of a patient with an ovarianneoplasm (Mountford, et al., Proteolipid identified by magneticresonance spectroscopy in plasma of a patient with borderline ovariantumor. Lancet 1:829-834). While further evaluation of the proteolipidusing the orcinol method suggested RNA was present, this could not beconfirmed using other methods. Wieczorek and associates, using UVspectrometry and hydrolysis by RNases, claimed to have found a specificRNA-proteolipid complex in the serum of cancer patients which was notpresent in healthy individuals (Wieczorek et al., 1085, Isolation andcharacterization of an RNA-proteolipid complex associated with themalignant state in humans. Proc Natl Acad Sci USA 82:3455-3459;Wieczorek et al., 1987, Diagnostic and prognostic value ofRNA-proteolipid in sera of patients with malignant disorders followingtherapy: First clinical evaluation of a novel tumor marker. Cancer Res47:6407-6412). The complex had unvarying composition regardless of thetype cancer. Wieczorek et al. were further able to detect this specificRNA-proteolipid complex using a phage DNA cloned into E. coli andhybridized to RNA from neoplastic serum, a method distinctly differentfrom the method of this invention. The DNA was then detected byimmunoassay (Wieczorek & Rhyner, 1989, Ein gesondentest furRNA-proteolipid in serumproben bei neoplasie. Schweiz med Wschr119:1342-1343). However, the RNA found in this complex is described as10 kilobases, which is so large as to make it questionable whether thistruly represents RNA as described. More recently, DNA and RNA-containingnucleoprotein complexes, possibly representing functional nuclearsuborganellular elements, were isolated from the nuclei of lymphomacells (Rosenberg-Nicolson & Nicolson, 1992, Nucleoprotein complexesreleased from lymphoma nuclei that contain the abl oncogene and RNA andDNA polymerase and RNA primase activities. J Cell Biochem 50:43-52). Itwas not shown, however, that these complexes can be shedextracellularly. Other ribonucleoprotein complexes have been associatedwith c-myc oncogene RNA (Chu et al., 1995, Thymidylate synthase binds toc-myc RNA in human colon cancer cells and in vitro. Mol. Cell. Biol.15:179-185).

While plasma and serum are generally presumed to be cell-free, in thepractical sense, particularly under conditions of routine clinicalfractionation, plasma and serum may occasionally be contaminated bycells. These contaminating cells are a source of intracellular RNA whichis detectable by the methods of the invention. While the level ofcontaminating cells may be reduced by filters or high speedcentrifugation, these methods may also reduce extracellular RNA,particularly larger apoptotic bodies. Clinical utility of the inventionis not dependent upon further separating of plasma or serum RNA into itsextracellular and intracellular species.

Similar analogy likely exists for detection of normal RNA (non-tumorderived or non-tumor associated RNA) in plasma and serum. Subsequent tothe filing of the provisional patent application for this patent, theinventor has shown that normal RNA (non-tumor derived RNA) couldsimilarly be detected in the plasma or serum of both healthy volunteersand cancer patients using extraction methods and amplification methodsas described by this invention. Qualitative results suggested thatamplified product was greater when obtained from cancer patients.Further, use of a 0.5 micron filter prior to amplification reduced, butdid not eliminate amplifiable RNA, consistent with extracellular RNAbeing of variable size, with additional contaminating cells possible.

While the methods of RNA extraction utilized in this invention have beenpreviously used to extract both viral RNA and intracellular RNA, theirapplicability to extracellular tumor-related or tumor-associated RNA wasnot obvious. The physical characteristics of the extracellular RNAcomplexes remain largely unknown, and thus it was not known prior tothis invention if the methods of extraction to be described couldeffectively remove extracellular RNA from their proteo-lipid, apoptotic,vesicular, or protein-bound complexes.

This invention describes the applicability of these RNA extractionmethods to the extraction of extracellular RNA from plasma or serum, andthus describes a new use for these extraction methods.

In summary, this invention describes a method by which RNA in plasma orserum can be detected and thus utilized for the detection, monitoring,or evaluation of cancer or premalignant conditions. This method utilizesnucleic acid amplification assays to detect human or animaltumor-derived or associated extracellular RNA circulating in plasma orserum. It also enables extraction and amplification of intracellular RNAshould cells be present in plasma or serum. The described extractionmethods and various nucleic acid amplification assays, including but notlimited to RT-PCR, branched DNA signal amplification, transciption-basedamplification, amplifiable RNA reporters, boomerang DNA amplification,strand displacement activation, cycling probe technology, isothermalNASBA amplification, and other self-sustained sequence replicationassays, have not been used for the detection of tumor-derived ortumor-associated RNA in plasma or serum, reflecting the generalscientific bias that mammalian extracellular RNA does not existcirculating in plasma or serum, despite isolated studies to thecontrary. Thus, this invention represents both a novel and non-obviousmethod of detecting, monitoring, and evaluating cancer or premalignantconditions, and a novel and non-obvious application of both RNAextraction methodology and nucleic acid amplification assays. Thisinvention, as described below entails a multi-step procedure applied toplasma or serum which consists of three parts, with the initial step(Part A) involving extraction of tumor-derived or associated RNA fromplasma or serum, a second step (Part B) involving application of anucleic acid amplification assay, in which reverse transciption of RNAto its cDNA may be involved, and a third step (Part C) involvingdetection of the amplified product. Any nucleic acid amplification assaycapable of permitting detection of small numbers of RNA molecules ortheir corresponding cDNA may be used in Part B. Similarly, variousmethods of detection of amplified product may be used in Part C,including but not limited to agarose gel electrophoresis, ELISAdetection methods, electrochemiluminescence, high performance liquidchromatography, and reverse dot blot methods. Furthermore, Part B andPart C may utilize assays which enable either qualitative orquantitative RNA analysis. Thus, while this invention uses variousmethods described in the literature, it is the unique application ofthese methods to the detection of tumor-derived or associatedextracellular RNA from plasma or serum that makes this invention novel.This invention provides a simple means for testing blood plasma or serumfor tumor-derived or associated RNA, with the result of identifyingpatients harboring tumor cells. Since this invention enables detectionof extracellular RNA, and does not depend upon the presence ofcirculating cancer cells, it offers a sensitive yet inexpensive screenfor both malignancy and pre-malignancy, as well as a way for monitoringcancer and obtaining other prognostically important clinicalinformation.

OBJECTS AND APPLICATIONS OF THE INVENTION

It is therefore the object of this invention to detect or infer thepresence of cancerous or precancerous cells whether from non-hematologicor hematologic malignancy, within a human or animal body, both in thoseknown to have cancer and in those not previously diagnosed, by examiningthe plasma or serum fraction of blood for tumor-derived or associatedextracellular RNA, including, but not limited to, that derived frommutated oncogenes, using nucleic acid amplification assays, such as, butnot limited to, polymerase chain reaction (RT-PCR), branched DNA signalamplification, isothermal nucleic acid sequence based amplification(NASBA), other self-sustained sequence replication assays,transcription-based amplification, boomerang DNA amplification, stranddisplacement activation, cycling probe technology, and amplifiable RNAreporters.

An application of this invention is to allow identification or analysis,either quantitatively or qualitatively, of tumor-derived or associatedRNA in the blood plasma or serum of humans or animals during orfollowing surgical procedures to remove premalignant or malignantlesions, and thus to allow stratification of such patients as to theirrisk of residual cancer following the surgery.

Another application of this invention is to allow identification oranalysis, either quantitatively or qualitatively, of tumor-derived orassociated RNA in the blood plasma or serum of humans or animals who arereceiving cancer therapies, including but not limited to biotherapy,chemotherapy, or radiotherapy, as a guide to whether adequatetherapeutic effect has been obtained or whether additional oralternative therapy is required, and further, to assess prognosis inthese patients.

Another application of this invention is to allow identification oranalysis, either quantitatively or qualitatively, of tumor-derived orassociated RNA in the blood plasma or serum of humans or animals whohave completed therapy as an early indicator of relapsed cancer,impending relapse, or treatment failure.

Another application of this invention is to allow identification, eitherby detection or by inference, of the presence of premalignant neoplasmsincluding dysplasias or adenomas by the examination of blood plasma orserum for RNA derived from or associated with those neoplasms.Furthermore, analysis, for example by a panel of assays to detectvarious RNA, may serve to distinguish malignant from premalignantconditions, or assist in medical monitoring to detect is transformationof a neoplasm to an outright malignancy, or to detect regression.

Thus, an application of this invention is to provide a method ofscreening both individuals without known risk, and individuals at risk,for cancer and premalignant conditions, and further, for defining riskof cancer when that risk is unknown.

Another application of this invention is to allow identification oranalysis, either quantitatively or qualitatively, of tumor-derived orassociated RNA in the blood plasma or serum of humans or animals eithernewly or recently diagnosed with cancer or a premalignant condition inorder to clarify when to initiate therapy, including adjuvant therapies.

Another application of this invention is to allow identification oranalysis of tumor-derived or associated RNA, either singularly or by apanel approach detecting varied RNA, in the blood plasma or serum ofhumans or animals in order to determine specific characteristics of agiven patient's tumor, as to assist in the development ofpatient-specific therapies, help direct a given patient into a giventreatment regimen, or help predict prognosis or tumor behavior.

SUMMARY OF THE INVENTION

The objects, advantages and applications of the present invention areachieved by the hereinafter described method for detecting tumor derivedor associated extracellular RNA from body fluids, in particular frommammalian blood plasma or serum by (A) extraction of RNA from bloodplasma or serum; (B) amplification of the RNA by nucleic acidamplification assays, including (1) reverse transcription polymerasechain reaction (RT-PCR), ligase chain reaction, branched DNA signalamplification, transcription-based amplification, amplifiable RNAreporters, Q-beta replication, boomerang DNA amplification, stranddisplacement activation, cycling probe technology, isothermal nucleicacid sequence based amplification (NASBA) and self-sustained sequencereplication assays. The primers used may be selected for their abilityto characterize the tumor; and (C) detection of the specific amplifiedRNA.

This method of detection can be employed in various methods of useincluding the detection of early cancers and premalignant neoplasms andinvasive or advanced cancers, and for the monitoring of patients duringtreatment therapy and for post-operative monitoring, and to developappropriate patient-specific treatment strategies as described herein.

DETAILED DESCRIPTION OF THE INVENTION

The use of RNA detection is preferred in many circumstances over DNAdetection since a greater number of RNA molecules are potentiallyavailable, thus allowing potentially greater sensitivity. Furthermore,since wild-type DNA genetic information is identical in all somaticcells of an individual, discrimination between normal andtumor-associated DNA is dependent upon the presence of a mutation.Detection of RNA, by reflecting activity of the gene, allowsdemonstration of an inappropriately expressing non-mutated gene, as istypically seen in malignancy. Thus, RNA amplification methods allow away to detect gene expression, whether normal or mutated, which isturned on in cancer. The present invention provides a much greaterapplicability and versatility to monitoring cancer than do any methodsbased on DNA analysis. For a DNA method to detect cancers from normals,there must be some mutation or genetic rearrangement present in thecancer, but not in the normal. The present process of using RNA willsimilarly detect the mutant RNA produced from this DNA. However, itfurther allows detection of inappropriately expressing “normal” genes.Thus, compared to methods detecting DNA, methods detecting RNA providegreater versatility and applicability in addition to the expectedgreater sensitivity.

This invention relates to a method of detecting or inferring thepresence of cancerous or precancerous cells, whether from anon-hematologic malignancy (i.e., solid tumor) or from a hematologicmalignancy, in a human or animal by the combination of three stepsapplied to plasma or serum. The first step (Part A) involves theextraction of tumor-derived or associated RNA from blood plasma orserum. The second step (Part B) applies a nucleic acid amplificationassay to the extracted RNA. In this step, the extracted RNA may first bereverse transcribed to cDNA prior to amplification of the cDNA. Thethird step (Part C) allows for the detection of the amplified product.Parts B and C may be performed as to allow either qualitative orquantitative detection of the RNA, depending upon the ultimate clinicalobjective or application, as described herein. Various methods, asdetailed below, may be used in Part A. Similarly, any nucleic acidamplification assay which can be utilized in the detection of smallnumbers of RNA or corresponding cDNA molecules, including but notlimited to the polymerase chain reaction (RT-PCR), branched DNA signalamplification, ligase chain reaction, isothermal nucleic acid sequencebased amplification (NASBA), Q-beta replication, transcription-basedamplification, amplifiable RNA reporters, boomerang DNA amplification,strand displacement activation, cycling probe technology, and otherself-sustained sequence replication assays, as well as variations onthese including methods for nucleic acid enrichment such as by usingrestriction digestion with polymerase chain reaction and the use ofnested primers, may be used in Part B. Similarly, any method capable ofdemonstrating amplified nucleic acid product, including but not limitedto agarose gel electrophoresis, ELISA detection methods,electrochemiluminescence, high performance liquid chromatography, andreverse dot blot methods, may be used in Part C. In this invention, anyof the various methods in Part A may be combined with any methodapplicable for Part B, which can then be combined with any applicablemethod in Part C. It is the new application of these methods to thedetection of tumor-derived or associated RNA in plasma or serum, and inparticular to extracellular RNA but also to plasma or serumintracellular RNA, that makes this invention novel. Several methodsapplicable for each of Part A, Part B, and Part C, will be described indetail below as a description of the invention. Again, it is to beemphasized that any method in Part A can be combined with any method inPart B, with any method in Part C to follow. Furthermore, it should beemphasized that while the contribution of extracellular RNA versusintracellular RNA as detected in plasma or serum may be defined, forexample by using filters or high speed centrifugation, it is not arequirement of the invention that such a definition be made.

Either “fresh” blood plasma or serum, or frozen (stored) andsubsequently thawed plasma or serum may be used for purposes of thisinvention. Frozen (stored) plasma or serum should optimally bemaintained at storage conditions of −20 to −70 degrees centigrade untilthawed and used. “Fresh” plasma or serum should be refrigerated ormaintained on ice until used, with RNA extraction being performed assoon as possible.

Blood is drawn by standard methods into a collection tube, preferablysiliconized glass, either without anticoagulant for preparation ofserum, or with EDTA, sodium citrate, heparin, or similar anticoagulantsfor preparation of plasma. The preferred method if preparing plasma orserum for storage, although not an absolute requirement, is that plasmaor serum be first fractionated from whole blood prior to being frozen.This reduces the burden of extraneous intracellular RNA released fromlysis of frozen and thawed cells which might reduce the sensitivity ofthe amplification assay or interfere with the amplification assaythrough release of inhibitors to PCR such as porphyrins and hematin.“Fresh” plasma or serum may be fractionated from whole blood bycentrifugation, using preferably gentle centrifugation at300-800.times.g for five to ten minutes, or fractionated by otherstandard methods. High centrifugation rates capable of fractionating outapoptotic bodies should be avoided. Since heparin may interfere withRT-PCR, use of heparinized blood may require pretreatment withheparinase as described (Imai et al., 1992, Detection of HIV-1 RNA inheparinized plasma of HIV-1 seropositive individuals. J Virol Methods36:181-184), followed by removal of calcium prior to reversetranscription, as described (Id.). Thus, EDTA is the preferredanticoagulant for blood specimens in which PCR amplification is planned.

Part A: Extraction of Extracellular RNA from Plasma or Serum

In Part A, RNA extraction methods previously published for theextraction of mammlian intracellular RNA or viral RNA may be adapted,either as published or with modification, for extraction oftumor-derived or associated RNA from plasma and serum. The volume ofplasma or serum used in part A may be varied dependent upon clinicalintent, but volumes of 100 microliters to one milliliter of plasma orserum are sufficient in part A, with the larger volumes often indicatedin settings of minimal or premalignant disease. For example:

Both extracellular RNA and intracellular RNA may be extracted fromplasma or serum using silica particles, glass beads, or diatoms, as inthe method or adaptations of Boom et al. (1994, Rapid and simple methodfor purification of nucleic acids. J Clin Micro 28:495-503). Applicationof the method adapted by Cheung et al. (1994, Rapid and sensitive methodfor detection of hepatitis C virus RNA by using silica particles. J ClinMicro 32:2593-2597) is described:

Size fractionated silica particles are prepared by suspending 60 gramsof silicon dioxide (SiO₂, Sigma Chemical Co., St. Louis, Mo.) in 500milliliters of demineralized sterile double-distilled water. Thesuspension is then settled for 24 hours at room temperature.Four-hundred thirty (430) milliliters of supernatant is removed bysuction and the particles are resuspended in demineralized, steriledouble-distilled water added to equal a volume of 500 milliliters. Afteran additional 5 hours of settlement, 440 milliliters of the supernatantis removed by suction, and 600 microliters of HCl (32% wt/vol) is addedto adjust the suspension to a pH2. The suspension is aliquotted andstored in the dark.

Lysis buffer is prepared by dissolving 120 grams of guinidinethiocyanate (GuSCN, Fluka Chemical, Buchs, Switzerland) into 100milliliters of 0.1 M Tris hydrochloride (Tris-HCl) (pH 6.4), and 22milliliters of 0.2 M EDTA, adjusted to pH 8.0 with NaOH, and 2.6 gramsof Triton X-100 (Packard Instrument Co., Downers Grove, Ill.). Thesolution is then homogenized.

Washing buffer is prepared by dissolving 120 grams of guinidinethiocyanate (GuSCN) into 100 milliliters of 0.1 M Tris-HCl (pH 6.4).

One hundred microliters to two hundred fifty microliters (with greateramounts required in settings of minimal disease) of plasma or serum aremixed with 40 microliters of silica suspension prepared as above, andwith 900 microliters of lysis buffer, prepared as above, using anEppendorf 5432 mixer over 10 minutes at room temperature. The mixture isthen centrifuged at 12,000.times.g for one minute and the supernatantaspirated and discarded. The silica-RNA pellet is then washed twice with450 microliters of washing buffer, prepared as above. The pellet is thenwashed twice with one milliliter of 70% (vol/vol) ethanol. The pellet isthen given a final wash with one milliliter of acetone and dried on aheat block at 56 degrees centigrade for ten minutes. The pellet isresuspended in 20 to 50 microliters of diethyl procarbonate-treatedwater at 56 degrees centigrade for ten minutes to elute the RNA. Thesample can alternatively be eluted for ten minutes at 56 degreescentigrade with a TE buffer consisting of 10 millimolar Tris-HCl-onemillimolar EDTA (pH 8.0) with an RNase inhibitor (RNAsin, 0.5U/microliter, Promega), with or without Proteinase K (100 ng/ml) asdescribed by Boom et al. (1991, Rapid purification of hepatitis B virusDNA from serum. J Clin Micro 29:1804-1811). Following elution, thesample is then centrifuged at 12,000.times.g for three minutes, and theRNA containing supernatant recovered. The RNA extract is now used inPart B.

As an alternative method, both extracellular RNA and intracellular RNAmay be extracted from plasma or serum in Part A using the AcidGuanidinium Thiocyanate-Phenol-Chloroform extraction method described byChomozynski and Sacchi (1987, Single-step method of RNA isolation byacid guanidinium thiocyanate-phenol-chloroform extraction. AnalyticalBiochemistry 162:156-159) as follows:

The denaturing solution consists of 4 M guanidinium thiocyanate, 25millimolar sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M2-mercaptoethanol. The denaturing solution is prepared as follows: Astock solution is prepared by dissolving 250 grams of guanidiniumthiocyanate (GuSCN, Fluka Chemical) with 293 milliliters ofdemineralized sterile double-distilled water, 17.6 milliliters of 0.75 Msodium citrate, pH 7.0, and 26.4 milliliters of 10% sarcosyl at 65degrees centigrade. The denaturing solution is prepared by adding 0.36milliliters 2-mercaptoethanol/50 milliliters of stock solution.

One hundred microliters to one milliliter of plasma or serum is mixedwith one milliliter of denaturing solution. Sequentially, 0.1 milliliterof 2 N sodium acetate, pH 4.0, 1 milliliter of phenol, and 0.2milliliter of chloroform-isoamyl alcohol (49:1) are added, with mixingafter addition of each reagent. The resultant mixture is shakenvigorously for 10 seconds, cooled on ice for 15 minutes, and thencentrifuged at 10,000.times.g for 20 minutes at 4 degrees centigrade.The aqueous phase is then transferred to a clean tube and mixed with 1milliliter of isopropanol. The mixture is then cooled at −20 degreescentigrade for 1-2 hours to precipitate RNA. After centrifugation at10,000.times.g for 20 minutes the resulting RNA pellet is dissolved in0.3 milliliter of denaturing solution, and then reprecipitated with 1volume isopropanol at −20 degrees centigrade for one hour. Followinganother centrifugation at 10,000.times.g for ten minutes at 4 degreescentigrade, 75% ethanol is added to resuspend the RNA pellet, which isthen sedimented and vacuum dried, and then dissolved in 5-25 microlitersof 0.5% SDS at 65 degrees centigrade for ten minutes. The RNA extract isnow used in Part B.

As the preferred embodiment for Part A, and as an alternative method,extracellular RNA and intracellular RNA may be extracted from plasma orserum in Part A using variations of the acid guanidiniumthiocyanate-phenol-chloroform extraction method. For example, in thepreferred embodiment RNA is extracted from plasma or serum using TRIreagent, a monophase guanidine-thiocyanate-phenol solution, as describedby Chomczynski (1993, A reagent for the single-step simultaneousisolation of RNA, DNA and proteins from cell and tissue samples. Biotech15:532-537). One hundred microliters to one milliliter of plasma orserum is processed using one milliliter of TRI Reagent™ (TRI Reagent,Sigma Trisolv™, BioTecx Laboratories, Houston, Tex., TRIzol™, GIBOOBRL/Life Technologies, Gaithersburg, Md., ISOGEN™), Nippon Gene, Toyama,Japan, RNA Stat™60, Tel-test, Friendsword, Tex.) according tomanufacturer's directions. Minor adaptations may be applied as currentlypracticed within the art. Thus, from one hundred microliters to onemilliliter of plasma or serum is mixed with one milliliter of TRIReagent. Then 0.2 milliliter of chloroform is mixed for 15 seconds, andthe mixture allowed to stand for 3 minutes at room temperature. Themixture is then centrifuged at 4 degrees centigrade for 15 minutes at12,000.times.g. The upper aqueous phase is removed to which 0.5milliliter of isopropanol is mixed, and then left at room temperaturefor five minutes, followed by centrifugation at 4 degrees centigrade forten minutes at 12,000.times.g. The RNA pellet is then washed with onemilliliter of 75% ethanol by centrifuging at 12,000.times.g for 5minutes. The pellet is air dried and resuspended in 11.2 microliters ofRNAse free water. Contamination by polysaccharides and proteoglycans,which may be present in extracellular proteolipid-RNA complexes, may bereduced by modification of the precipitation step of the TRI Reagent™procedure, as described by Chomczynski and Mackey (1995, Modification ofthe TRI reagenttm procedure for isolation of RNA from polysaccharide-and proteoglycan-rich sources. BioTechniques 19:942-945) as follows:

One hundred microliters to one milliliter of plasma or serum is mixedwith TRI Reagent™ as per manufacturer's directions, being subjected tophase separation using either chloroform or bromo-cholorpropane(Chomczynski & Mackey, 1995, Substitution of choroform bybromo-chloropropane in the single-step method of RNA isolation.Analytical Biochemistry 225:163-164) and centrifugation at10,000.times.g for 15 minutes. The aqueous phase is removed and thenmixed with 0.25 milliliters of isopropanol followed with 0.25milliliters of a high-salt precipitation solution (1.2 M NaCl and 0.8 Msodium citrate). The mixture is centrifuged at 10,000.times.g for 5minutes and washed with one milliliter of 75% ethanol. The RNA pellet isthen vacuum dried and then dissolved in 5-25 microliters of 0.5% SDS at65 degrees centigrade for ten minutes. The RNA extract is now used inPart B.

Alternative methods may be used to extract RNA from plasma or serum inPart A, including but not limited to centrifugation through a cesiumchloride gradient, including the method as described by Chirgwin et al.(1979, Isolation of biologically active ribonucleic acid from sourcesenriched in ribonuclease. Biochemistry 18:5294-5299), andco-precipitation of extracellular RNA from plasma or serum with gelatin,such as by adaptations of the method of Fournie et al. (1986, Recoveryof nanogram quantities of DNA from plasma and quantitative measurementusing labeling by nick translation. Analytical Biochemistry 158:250-256)to RNA extraction.

Circulating extracellular deoxyribonucleic acid (DNA), includingtumor-derived or associated extracellular DNA, is also present in plasmaand serum (Stroun et al., 1989, Neoplastic characteristics of the DNAfound in the plasma of cancer patients. Oncology 46:318-322). Since thisDNA will additionally be extracted to varying degrees during the RNAextraction methods described above, it may be desirable or necessary(depending upon clinical objectives) to further purify the RNA extractand remove trace DNA prior to proceeding to Part B. This may beaccomplished using DNase, for example by the method as described byRashtchian (1994, Amplification of RNA. PCR Methods Applic 4:S83-S91),as follows:

For one microgram of RNA, in a 0.5 milliliter centrifuge tube placed onice, add one microliter of 10.times.DNase I reaction buffer (200micromolar Tris-HCl (pH 8.4), 500 micromolar KCl, 25 micromolar MgCl,one micromolar per milliliter BSA). Add to this one unit DNase I(GIBCO/BRL catalog #18068-015). Then bring the volume to ten microliterwith DEPC-treated distilled water, and follow by incubating at roomtemperature for 15 minutes. The DNase I is then inactivated by theaddition of 20 millimolar EDTA to the mixture, and heating for 10minutes at 65 degrees centigrade. The treated RNA may now go directly toPart B.

Alternatively, primers in Part B may be constructed which favoramplification of the RNA products, but not of contaminating DNA, such asby using primers which span the splice junctions in RNA, or primerswhich span an intron. Alternative methods of amplifying RNA but not thecontaminating DNA include the methods as described by Moore et al.(1991, Design of PCR primers that detect only mRNA in the presence ofDNA. Nucleic Acids Res. 18:1921), and methods as described by Buchman etal. (1993, Selective RNA amplification: A novel method usingdUMP-containing primers and uracil DNA glycosylase. PCR Methods Applic3:28-31), which employs a dU-containing oligonucleotide as an adaptorprimer.

Part B: Nucleic Acid Amplification

In Part B, RNA which has been extracted from plasma or serum during PartA, or its corresponding cDNA, is amplified using any nucleic acidamplification assay utilized for detection of low numbers of RNAmolecules. Applicable assays include but are not limited to reversetranscriptase polymerase chain reaction (RT-PCR), ligase chain reaction(Abravaya et al., 1995, Detection of point mutations with a modifiedligase chain reaction (Gap-LCR). Nucleic Acids Research 23:675-682),branched DNA signal amplification (Urdea et al., 1993, Direct andquantitative detection of HIV-1 RNA in human plasma with a branched DNAsignal amplification assay. AIDS 7(suppl2):S11-S14), amplifiable RNAreporters, Q-beta replication, transcription-based amplification,boomerang DNA amplification, strand displacement activation, cyclingprobe technology, isothermal nucleic acid sequence based amplification(NASBA) (Kievits et al., 1991, NASBA.TM.isothermal enzymatic in vitronucleic acid amplification optimized for the diagnosis of HIV-1infection. J Virological Methods 35:273-286), and other self-sustainedsequence replication assays. It is not necessary to modify these assaysfrom their published methods for Part B. The referenced publications areincorporated herein by reference in their entirety for theirdescriptions for performing the various assays identified therein. It isthe application of these nucleic acid amplification assays to thedetection of tumor-derived or associated extracellular RNA in plasma orserum that makes their use novel. The preferred embodiment for Part Buses the reverse transcriptase polymerase chain reaction (RT-PCR).

Primers used in the amplification assay should be based on the specifictumor-derived or associated RNA of interest which characterizes thetumor. Tumor-derived or associated RNA includes but is not limited to:

mRNA related to mutated oncogenes or mutated DNA, a partial list ofwhich includes H-ras, K-ras, N-ras, c-myc, her-2-neu, bcr-abl, fms, src,fos, sis, jun, erb-B-1, VHL, PML/RAR, AML1-ETO, EWS/FLI-1, EWS/ZRG.

mRNA related to tumor suppressor genes, a partial list of which includesp53, RB, MCC, APC, DCC, NF1, WT.

mRNA related to tumor-associated protein which is found elevated incertain cancers, a partial list of which includes alpha-feto protein(AFP), carcinoembryonic antigen (CEA), TAG-72, CA 19-9, CA-125, prostatespecific antigen (PSA), CD44, and hcg (human chorionic gonadotropin).

mRNA related to tumor-derived protein not normally found circulating inblood, a partial list of which includes tyrosinase mRNA, keratin 19mRNA.

mRNA related to tumor-specific antigens, such as in MAGE 1, MAGE 2, MAGE3, MAGE 4, GP-100, and MAGE 6, NUC 18, P97.

mRNA or messenger-like RNA associated with ribonucleoproteins and RNAwithin ribonucleoproteins, a partial list of which includes telomeraseRNA, and RNA associated with heterogenous nuclear ribonucleoprotein A1(hn RNP-A1) and A2/B1 (hn RNP-A2/B1) complexes, and heterogenous nuclearribonucleoprotein K (hn RNP-K), such as c-myc oncogene RNA, in additionto those RNA previously described above when associated withribonucleoprotein.

For example, oligonucleotide primer sequences for the bcr-abl transcriptmay be as follows (Edmands et al., 1994, Rapid RT-PCR amplification fromlimited cell numbers. PCR Methods Applic 3:317-319): Primer 1 at theM-bcr location: (5′-TGGAGCTGCAGATGCTGACCAACTCG-3′). Primer 2 at the exonII abl location: (5′-ATCTCCACTGGCCACAAAATCATACA-3′). Primer 3 at theM-bcr location: (5′-GAAGTGTTTCAGAAGCTTCTCC-3′). Primer 4 at the exon IIabl location: (5′-TGATTATAGCCTAAGACCCGGA-3′).

The nested RT-PCR assay yields a 305 or a 234 base pair product,depending upon bcr exon 3 expression.

As another example, nested primers for human tyrosinase cDNAamplification can be as follows (Brossart et al., 1994, Detection ofresidual tumor cells in patients with malignant melanoma responding toimmunotherapy. J. Immunotherapy 15:38-41): Primer 1 (outer, sense) -(5′-TTGGCAGATTGTCTGTAGCC-3′) Primer 2 (outer, anti-sense) -(5′-AGGZATTGTGCATGCTGZTT-3′) Primer 3 (nested, sense) -(5′-GTCTTTATGCAATGGAACGC-3′) Primer 4 (nested, anti-sense) -(5′-GCTATCCCAGTAAGTGGACT-3′).

The outer primers result in a PCR amplification product of 284 basepairs, and the nested primers result in a fragment of 207 base pairs.

The preferred oligonucleotide primer sequences for specifictumor-related or tumor-associated mRNA are previously published, withreferenced publications incorporated herein by reference in theirentirety.

Some, but not all, amplification assays require reverse transcription ofRNA to cDNA. As noted, the method of reverse transcription andamplification may be performed by previously published or recommendedprocedures, which referenced publications are incorporated herein byreference in their entirety, and modification is not required by theinvention beyond steps as described in Part A. Various reversetranscriptases may be used, including, but not limited to, MMLV RT,RNase H.sup.-mutants of MMLV RT such as SuperScript and SuperScript II(Life Technologies, GIBCO BRL, Gaithersburg, Md.), AMV RT, andthermostable reverse transcriptase from Thermus Thermophilus. Forexample, one method, but not the only method, which may be used toconvert RNA extracted from plasma or serum in Part A to cDNA is theprotocol adapted from the Superscript II Preamplification system (LifeTechnologies, GIBCO BRL, Gaithersburg, Md.; catalog no. 18089-011), asdescribed by Rashtchian (1994, Amplification of RNA. PCR Methods Applic4:S83-S91), adapted as follows:

1-5 micrograms of RNA extracted from plasma or serum in Part A in 13microliters of DEPC-treated water is added to a clean microcentrifugetube. Then one microliter of either oligo (dT) (0.5milligram/milliliter) or random hexamer solution (50 ng/microliter) isadded and mixed gently. The mixture is then heated to 70 degreescentigrade for 10 minutes and then incubated on ice for one minute.Then, it is centrifuged briefly followed by the addition of 2microliters of 10.times.synthesis buffer (200 mM Tris-HCl, pH 8.4, 500mM KCl, 25 mM magnesium chloride, one milligram/milliliter of BSA), onemicroliter of 10 mM each of dNTP mix, 2 microliters of 0.1 M DTT, onemicroliter of SuperScript II RT (200 U/microliter) (Life Technologies,GIBCO BRL, Gaithersburg, Md.). After gentle mixing, the reaction iscollected by brief centrifugation, and incubated at room temperature forten minutes. The tube is then transferred to a 42 degrees centigradewater bath or heat block and incubated for 50 minutes. The reaction isthen terminated by incubating the tube at 70 degrees centigrade for 15minutes, and then placing it on ice. The reaction is collected by briefcentrifugation, and one microliter of RNase H (2 units) is addedfollowed by incubation at 37 degrees centigrade for 20 minutes beforeproceeding to nucleic acid amplification.

Nucleic acid amplification then proceeds as follows:

To the cDNA mixture add the following: 8 microliters of10.times.synthesis buffer (200 mM Tris-HCl, pH 8.4, 500 mH KCl, 25 mMmagnesium chloride, 1 mg/ml of BSA), 68 microliters steriledouble-distilled water, one microliter amplification primer 1 (10micromolar), one microliter amplification primer 2 (10 micromolar), onemicroliter Taq DNA polymerase (2-5 U/microliter). Mix gently and overlaythe reaction mixture with mineral oil. The mixture is heated to 94degrees centigrade for 5 minutes to denature remaining RNA/cDNA hybrids.PCR amplification is then performed in an automated thermal-cycler for15-50 cycles, at 94 degrees centigrade for one minute, 55 degreescentigrade for 30 to 90 seconds, and 72 degrees centigrade for 2minutes. The amplified PCR product is then detected in Part C.

Furthermore, if the primers contain appropriate restriction sites,restriction digestion may be performed on the amplified product to allowfurther discrimination between mutant and wild-type sequences.

Cycling parameters and magnesium concentration may vary depending uponthe specific case. For example, an alternative method using nestedprimers useful for detection of human tyrosinase mRNA in Part B is themethod described by Smith et al. (1991, Detection of melanoma cells inperipheral blood by means of reverse transcriptase and polymerase chainreaction. Lancet 338:1227-1229), as follows:

Primer sequences are as described above for human tyrosinase. Tenmicroliters of RNA extracted in Part A from plasma or serum are treatedfor reverse transcription by heating at 90 degrees centigrade for 4minutes, cooling rapidly, and diluting to 20 microliters with a mixtureconsisting of 1.times.PCR buffer (10 mmol/liter Tris-HCl, pH 8.4, 50mmol/liter KCl, 100 microgram/milliter gelatin), 8 mmol/liter magnesiumchloride, 1 mmol/liter each DATP, dCTP, dGTP, and dTTP, 25 μmoltyrosinase primer 2 (as previously described), 20 units of ‘RNA guard’(Pharmacia), and 4 units of murine moloney leukemia virus reversetranscriptase (Pharmacia). The total mixture is then incubated at 37degrees centigrade for one hour, half the sample removed, and diluted to50 microliters containing 1.times.PCR buffer, 200 micromol/liter each ofdATP, dCTP, dGTP, and dTTP, 1.6 mmol/liter magnesium chloride, 150 μmolprimer 1 and primer 2, 0.1% Triton X-100, and 1 unit Taq DNA polymerase(Promega). The mixture is overlaid with oil, and heated at 95 degreescentigrade for 5 minutes, followed by 30 cycles of PCR in a thermalcycler at 95 degrees centigrade for 65 seconds, 55 degrees centigradefor 65 seconds, and 72 degrees centigrade for 50 seconds. The productsare then reamplified with nested primer 3 and nested primer 4 using 5microliters in a 1:100 dilution. These were amplified in a 25 microliterreaction volume for an additional 30 cycles. This final amplified PCRproduct is now detected in Part C, either by being electrophoresed on anagarose gel, or by other method.

The preferred embodiments for Part B amplification of specifictumor-related or tumor-associated RNA, including specific primers,method of reverse transciption, and method of RT-PCR, are described bythe following referenced publications which are incorporated herein byreference in their entirety for their description for performing thevarious assays identified therein.

For Part B amplification of tyrosinase mRNA, a mRNA associated withmalignant melanoma, the preferred method is that of Brossart et al.(1994, Detection of residual tumor cells in patients with malignantmelanoma responding to immunotherapy. J. Immunotherapy 15:38-41).

For Part B amplification of Keratin 19 mRNA, a mRNA associated withbreast cancer and other epithelial malignancies, the preferred method isthat of Datta et al. (1994, Sensitive detection of occult breast cancerby the reverse-transcriptase polymerase chain reaction. J Clin Oncol12:475-482).

For Part B amplification of prostate-specific antigen (PSA) mRNA, a mRNAassociated with prostate cancer, the preferred method is that of Katz etal. (1995, Enhanced Reverse Transcriptase-Polymerase Chain Reaction forProstate Specific Antigen as an Indicator of True Pathologic Stage inPatients with Prostate Cancer. Cancer 75:1642-1648).

For Part B amplification of alpha-fetoprotein (AFP) mRNA, a mRNAassociated with hepatocellular carcinoma, testicular cancer, and othercancers, the preferred method is that of Komeda et al. (1995, Sensitivedetection of circulating hepatocellular carcinoma cells in peripheralvenous blood. Cancer 75:2214-2219).

For Part B amplification of BCR/ab1 mRNA, a mRNA associated with chronicmysloid leukemia (CML), the preferred method is that of Stock et al.(1997, Value of molecular monitoring during the treatment of chronicmyeloid leukemia: A cancer and leukemia group B study. J. Clin. Oncology15:26-36), or alternatively, by the method of Edmonds et al. (1994,Rapid RT-PCR amplification from limited cell numbers. PCR Methods Applic3:317-319).

For Part B amplification of carcinoembryonic antigen (CEA) mRNA, a mRNAassociated with gastrointestinal cancers and breast cancer, thepreferred method is that of Gerhard et al. (1994, Specific detection ofcarcinoembryonic antigen-expressing tumor cells in bone marrow aspiratesby polymerase chain reaction. J. Clin. Oncol. 12:725-729).

For Part B amplification of P97 mRNA, a mRNA associated with malignantmelanoma, the preferred method is that of Hoon et al. (1995, Detectionof occult melanoma cells in blood with a multiple-marker polymerasechain reaction assay. J. Clin. Oncol. 13:2109-2116).

For Part B amplification of MUC 18 mRNA, a mRNA associated withmalignant melanoma, the preferred method is that of Hoon et al. (Id.).

For Part B amplification of PML/RAR-.alpha. mRNA, a mRNA associated withacute promyelocytic leukemia, the preferred method is that of Miller etal. (1993, Detection of minimal residual disease in acute promyelocyticleukemia by a reverse transcription polymerase chain reaction assay forthe PML/RAR-alpha fusion mRNA. Blood 82:1689-1694).

For Part B amplification of CD44 mRNA, a mRNA associated with lungcancer, the preferred method is that of Penno et al. (1994, Expressionof CD44 in human lung tumors. Cancer Research 54:1381-1387).

For Part B amplification of EWS/FLI-1 mRNA, a mRNA associated withEwing's sarcoma and other Ewing's tumors, the preferred method is thatof Pfleiderer et al. (1995, Detection of tumour cells in peripheralblood and bone marrow from Ewing tumour patients by RT-PCR. Int. J.Cancer (Pred. Oncol.) 64:135-139).

For Part B amplification of EWS/ERG mRNA, a mRNA associated with Ewing'ssarcoma and other Ewing's tumors, the preferred method is that ofPfleiderer et al. (Id.).

For Part B amplification of AML1/ETO mRNA, a mRNA associated with acutemyelogenous leukemia, the preferred method is that of Maruyama et al.(1994, Detection of AML1/ETO fusion transcript as a tool for diagnosingt(8; 21) positive acute myelogenous leukemia. Leukemia 8:40-45).

For Part B amplification of MAGE mRNA, including mRNA of MAGE-1, MAGE-2,MAGE-3, and MAGE-4, which are associated with bladder cancer, ovariancancer, melanoma, lung cancer, head and neck cancer, and others, thepreferred method is that of Patard et al. (1995, Expression of MAGEgenes in transitional-cell carcinomas of the urinary bladder. Int. J.Cancer 64:60-64).

For Part B amplification of beta-human chorionic gonadotropin mRNA, amRNA associated with malignant melanoma, germ cell tumors, and othercancers, the preferred method is that of Doi et al. (1996, Detection ofbeta-human chorionic gonadotropin mRNA as a marker for cutaneousmalignant melanoma. Int. J. Cancer 65:454-459).

For Part B amplification of human Telomerase-associated RNA, thepreferred method is by application of the TRAP PCR method as describedby Kim et al (1994, Specific association of human telomerase activitywith immortal cells and cancer. Science 266:2011-2015). Alternatively,other amplification methods may be used as described herein where primerselection is designed based upon the human Telomerase template sequenceas described by Feng et al (1995, The RNA component of human telomerase.Science 269:1236-1241).

Alternative methods of nucleic acid amplification which may be used inPart B include other variations of RT-PCR, including quantitativeRT-PCR, for example as adapted to the method described by Wang et al.(1989, Quantitation of mRNA by the polymerase chain reaction. Proc NatlAcad Sci USA 86:9717-9721) or by Karet et al. (1994, Quantification ofmRNA in human tissue using fluorescent nested reverse-transcriptasepolymerase chain reaction. Analytical Biochemistry 220:384-390).

An alternative method of nucleic acid amplification which may be used inPart B is ligase chain reaction (Sooknanan, R., Malek, L., Wang, X-H. etal.: Detection and direct sequence identification of BCR-ABL mRNA in PH+chronic myeloid leukemia. Experimental Hematology 21:1719-1724, 1993).Extracellular RNA extracted from plasma or serum in Part A must bereverse transcribed to cDNA. Oligonucleotide primers are selected whichlie directly upon the cDNA site of interest. If a mutation site ispresent, oligonucleotides which are complementary to the site are madeto contain the mutation only at their 3-prime end, excludinghybridization of non-mutated, wild-type DNA. Restriction sites can alsobe utilized to discriminate between mutant and wild-type sequences ifnecessary.

An alternative method of either qualitative or quantitativeamplification of nucleic acid which may be used in Part B is branchedDNA signal amplification, for example as adapted to the method describedby Urdea et al. (1993, Direct and quantitative detection of HIV-1 RNA inhuman plasma with a branched DNA signal amplification assay. AIDS7(suppl2):S11-S14), with modification from the reference as follows:plasma or serum should only be centrifuged at lower speeds, aspreviously outlined. Extracellular RNA is then extracted from plasma orserum as described in Part A, and then added directly to microwells. Themethod for detection of tumor-related or tumor-associated RNA thenproceeds as referenced (Id.), with target probes specific for thetumor-related or tumor-associated RNA or cDNA of interest, and withchemiluminescent light emission proportional to the amount oftumor-associated RNA in the plasma or serum specimen. The specifics ofthe referenced method are described further but Urdea et al. (1991,Branched DNA amplification multimers for the sensitive, direct detectionof human hepatitis viruses. Nucleic Acids Research Symposium Series24:197-200) with this reference incorporated herein in its entirety.

An alternative method of either qualitative or quantitativeamplification of nucleic acid which may be used in Part B is isothermalnucleic acid sequence based amplification (NASBA), for example asadapted to the method described by Kievits et al. (1991,NASBA.TM.isothermal enzymatic in vitro nucleic acid amplificationoptimized for the diagnosis of HIV-1 infection. J Virological Methods35:273-286), or by Vandamme et al. (1995, Detection of HIV-1 RNA inplasma and serum samples using the NASBA amplification system comparedto RNA-PCR. J Virological Methods 52:121-132). The method of Sooknananet al. (1995, Detection and direct sequence identification of BCR-ABLmRNA in Ph+ chronic myeloid leukemia. Experimental Hematology21:1719-1724) may be used for the detection and quantification ofBCR/ABL mRNA.

Alternative methods of either qualitative or quantitative amplificationof nucleic acids which may be used in Part B include, but are notlimited to, Q-beta replication, other self-sustained sequencereplication assays, transcription-based amplification assays, andamplifiable RNA reporters, boomerang DNA amplification, stranddisplacement activation, and cycling probe technology.

The amplified product from Part B is next detected in Part C. Dependingupon the detection method used in Part C, primers may need to bebiotinylated or otherwise modified in Part B.

Part C: Detection of Amplified Product

There are numerous methods to detect amplified nucleic acid product, anyof which may be used in Part C to detect the amplified product from PartB. The referenced publications, including those pertaining to detectionof specific tumor-related or associated RNA or its corresponding cDNA aspreviously cited, and those pertaining to RNA or its corresponding cDNAdetection as follows, are incorporated herein by reference in theirentirety for the descriptions for performing the various assaysidentified therein.

In the preferred method, amplified product is detected in Part C usinggel electrophoresis. In the preferred embodiment, 25 microliters ofamplified (or post-amplification digested) product is electrophoresedthrough a 3% agarose gel in 1.times.TBE at 75 VDC. Electrophoresis iscarried out for one to two hours before staining with ethidium bromide.As an alternative to ethidium bromide, the amplified product can betransferred from the gel to a membrane by blotting techniques to bedetected with a labeled probe (Nguyen, 1989, Southern blot analysis ofpolymerase chain reaction products on acrylamide gels. BioTechniques7:238-240).

An alternative method which may be used in Part C to detect theamplified product from Part B is ELISA detection. Depending upon theELISA detection method used, it may be necessary to biotinylate orotherwise modify the primers used in part B.

For example, one ELISA detection method which may be used in Part C isthe method described by Landgraf et al. (1991, Direct analysis ofpolymerase chain reaction products using enzyme-linked immunosorbentassay techniquest. Analytical Biochemistry 198:86-91), as follows:

Primers are modified with biotinylamidocaproat-N-hydroxysuccinimidester(Sigma) and fluoroescein isothiocyanate (FITC) (Sigma) by the method ofLandgraf et al. (1995, Quantitative analysis of polymerase chainreaction (PCR) products using primers labeled with biotin and afluorescent dye. Analytical Biochemistry 193:231-235). Followinginvention Part B, the ELISA is carried out in microtiter plates coatedwith 1 microgram/milliliter affinity-purified avidin (13 U/mg, Sigma).One microliter of the final amplification product (or post-digestionproduct) is diluted with 50 microliters of PBS-Tween, and then incubatedat room temperature for 30 minutes in the microtiter plate well.Non-incorporated primers are removed by washing with PBS-Tween. Theplates are then incubated at room temperature for 30 minutes afteradding 50 microliters per well of anti-FITC antibody-HRPO conjugate(Dakopatts) which is at a 1:500 dilution with PBS-Tween. Following this,80 microliters of an ELISA solution made from one milligram3,3′,5,5′-tetramethylbenzidin (Sigma) dissolved in one milliliterdimethyl sulfoxide, and diluted 1:10 with 50 millimol Na-acetate: citricacid, pH 4.9, with 3 microliter of 30% (vol/vol) water is added to eachwell. After 2-5 minutes, the reaction is stopped by adding 80 microliterof 2M H₂SO₄. The optical density is then read at 450 nm.

Alternative methods of ELISA detection which may be used in Part Cinclude, but are not limited to, immunological detection methods usingmonoclonal antibody specific for RNA/DNA hybrids, such as by adaptingmethods described by Coutlee et al. (1989, Immunodetection of DNA withbiotinylated RNA probes: A study of reactivity of a monoclonal antibodyto DNA-RNA hybrids. Analytical Biochemistry 181:96-105), or by Bobo etal. (1990, Diagnosis of chlamydia trachomatis cervical infection bydetection of amplified DNA with an enzyme immunoassay. J Clin Micro28:1968-1973), which publications are also incorporated herein byreference in their entirety for their description of the detectionmethods identified therein.

Alternative methods of ELISA detection which may be used in Part Cinclude, but are not limited to, commercial detection systems such asthe SHARP signal system (Digene Diagnostics, Inc.), and the DNA enzymeimmunoassay (DEIA), (GEN-ETI-K DEIA, Sorin Biomedica).

Alternative methods by which amplified product from Part B may bedetected in Part C include but are not limited to all methods ofelectrochemiluminescence detection, such as by adapting the methoddescribed by Blackburn et al. (1991, Electrochemiluminescence detectionfor development of immunoassays and DNA probe assays for clinicaldiagnostics. Clin Chem 37/9:1534-1539), or by DiCesare et al. (1993, Ahigh-sensitivity electrochemiluminescence-based detection system forautomated PCR product quantitation. BioTechniques 15:152-157), and allmethods utilizing reverse dot blot detection technology (Saiki et al.,1989, Genetic analysis of amplified DNA with immobilizedsequence-specific oligonucleotide probes. Science 233:1076-1078), andall methods utilizing high-performance liquid chromatography.

Therapeutic Applications

The extraction of extracellular tumor-associated or derived RNA fromplasma or serum, and the amplification of that RNA or its correspondingcDNA to detectable levels, permits further analysis or othermanipulation of that RNA, or the corresponding cDNA, from which furtherclinical utility is realized. In this optional step of the invention,amplified extracellular RNA or the corresponding cDNA is analyzed todefine the characteristics or composition of the tumor from which theRNA originates. Any of several methods may be used, dependent upon thedesired information, including nucleic acid sequencing, spectroscopyincluding proton NMR spectroscopy, biochemical analysis, and immunologicanalysis. In the preferred embodiment, amplified cDNA is isolated—forexample by excising DNA bands from an agarose gel—reamplified, clonedinto a plasmid vector, for example the pGEM-T vector plasmid (Promega)and sequenced using a commercial kit such as Sequenase 2.0 (USB).Analysis to define the characteristics or composition of thetumor-associated RNA in plasma or serum, and thus the tumor of origin,affords a wide array of clinical utility, including the description,characterization, or classification of the tumor, whether known oroccult, such as by tissue of origin, by type (such as premalignant ormalignant), phenotype, and genotype, and by description orcharacterization of tumor behavior, physiology and biochemistry, as togain understanding of tumor invasiveness, propensity to metastasize, andsensitivity or resistance to various therapies, thereby allowing theprediction of response to either ongoing or planned therapy and,further, allowing evaluation of prognosis. Comparison of thecharacteristics of extracellular RNA to previous biopsy or surgicalspecimens permits further evaluation of tumor heterogeneity orsimilarity in comparison to that specimen, and thus evaluation of tumorrecurrence.

Following extraction of extracellular tumor-derived or tumor-associatedRNA from plasma or serum and amplification of the corresponding cDNA,ribonucleic acid (RNA) may be transcribed or manufactured back from theamplified DNA as a further option. Transcription of RNA may be performedby employing a primer with an RNA polymerase promoter region joined tothe standard primer sequence of the cDNA in an amplification reaction.RNA is then transcribed from the attached promoter region. In thepreferred embodiment, amplified cDNA is cloned into an expressionvector, and RNA is transcribed. Furthermore, as an optional preferredembodiment, the RNA is used in an in vitro translation reaction tomanufacture tumor-associated or tumor-specific protein or associatedpeptides or oligopeptides, according to methods currently known in theart (Roggenbuck et al., 1991, Human Papillomavirus Type 18 E6 and E6,and E7 Protein Synthesis in Cell Free Translation Systems and Comparisonof E6 and E7 In Vitro Translation Products to ProteinsImmunoprecipitated from Human Epithelial Cells. J. Virol. 65:5068-72;.Dosaka et al., 1991, A Complex Pattern of Translational Initiation andPhosphorylation in L-Lyc Proteins. Oncogene 6:371-378; Alkema et al.,1993, Characterization and Chromosomal Localization of the HumanProto-Oncogene BMI-1. Human Mol. Genet. 2:1597-1603; Shen et al., 1995,Identification of the Human Prostate Carcinoma Oncogene PTI-1 by RapidExpression Cloning and Differential RNA Display. Proc. Natl. Acad. Sci.USA 92:6778-6782.). Note, these cited references, and those to follow,are incorporated herein by reference in their entirety for theirdescription for performing the various assays identified therein.

Extraction of tumor-derived or tumor-associated extracellular RNA, itsamplification, characterization, and translation to tumor-associated ortumor-specific protein, provides significant utility, both in theassignment of therapy and in the development of tumor-specifictherapies. Sequencing of RNA or cDNA allows assignment or development ofantisense compounds, including synthetic oligonucleotides and otherantisense constructs appropriately specific to the DNA, such as byconstruction of an expression plasmid such as by adapting the method ofAoki et al. (1995, Liposome-mediated in vivo gene transfer of antisenseK-ras construct inhibits pancreatic tumor dissemination in the murineperitoneal cavity. Cancer Res. 55:3810-3816) which is incorporated byreference in its entirety, or by other construction and use asreferenced (Cohen, 1991, Biochemical Therapy: Antisense Compounds. In:Biologic Therapy of Cancer, (DeVita V. T., Hellman S., Rosenberg, S. A.,eds.) J. B. Lippincott Co., Philadelphia, pp. 763-775; Polushin & Cohen,J1994, Antisense Pro-Drugs: 5-ester Oligodeoxynucleotides. Nucelic AcidsRes. 22:5492-5496; Sakakura et al., 1994, Inhibition of Gastric CancerCell Proliferation by Antisense Oligonucleotides Targeting the MessengerRNA Encoding Proliferating Cell Nuclear Antigen. Br. J. Cancer70:1060-1066; Colomeret al., 1994, erbB-2 Antisense OligonucleotidesInhibit the Proliferation of Breast Carcinoma Cells with erbB-2 OncogeneAmplification. Br. J. Cancer 70:819-825; Skorsk et al, 1994, Expressionof Philadelphial Leukemia Cell Growth in Mice by BCR-ABL AntisenseOligodeoxynucleotide. Proc. Natl. Acad. Sci. USA 91:4504-4508). Thus,application of the invention in this manner would entail the extractionof tumor-associated RNA from plasma or serum, followed by an optionalstep of reverse transcribing to cDNA, followed by amplification of theRNA or cDNA. The amplified product can then be sequenced to define thenucleic acid sequence of the tumor-associated RNA or cDNA. An antisenseoligonucleotide is then constructed in such a manner as referenced abovespecific to the defined sequence, or alternatively, an alreadymanufactured antisense compound is determined to be applicable, or maybe manufactured when the sequence is known based upon knowledge of theprimer sequence. Similarly, defining tumor characteristics by analysisof extracellular RNA allows assignment of specific monoclonal antibodyor vaccine therapies appropriately specific to the tumor. Production ofcorresponding immunologic protein can be used in the development oftumor-specific monoclonal antibodies. Thus, application of the inventionin this manner would entail the extraction of tumor-associated RNA fromplasma or serum, followed by amplification to obtain a tumor-associatedamplified product. The amplified product is translated, or transcribedand translated, into a protein or associated peptides or oligopeptidesas previously described, thus providing a tumor-associated antigen. Thetumor-associated antigen thus enables production of a monoclonalantibody directed against the antigen by use of hybridoma technology orother methods as currently practiced by the art (Schlom, 1991,Antibodies in Cancer Therapy: Basic Principles of Monoclonal Antibodies.In: Biologic Therapy of Cancer, (DeVita V. T., Hellman S., Rosenberg, S.A., eds.) J. B. Lippincott Co., Philadelphia, pp. 464-481). Saidmonoclonal antibody may further be conjugated with a toxin or othertherapeutic agent (Vitetta & Thorpe, 1991, Immunotoxins. In: BiologicTherapy of Cancer, (DeVita V. T., Hellman S., Rosenberg, S. A., eds.) J.B. Lippincott Co., Philadelphia), or with a radionucleotide (Larson etal., 1991, Radioisotope Conjugates. In: Biologic Therapy of Cancer,(DeVita V. T., Hellman S., Rosenberg, S. A., eds.) J. B. Lippincott Co.,Philadelphia, pp. 496-5114) to provide further therapeutic or diagnosticuse directed against the tumor. Similarly, translated protein orassociated peptides or oligopeptides can be used in tumor-specificvaccine development. Furthermore, the extracellular RNA andcomplimentary DNA permit a means of defining or allowing theconstruction of a DNA construct which may be used in vaccine therapy.Specifically, the invention is applied to either define or obtaintumor-associated protein or peptides, RNA, or cDNA, by methods aspreviously described, and from which a tumor-directed vaccine may bedeveloped or constructed. The methods by which the vaccine is furtherdeveloped or constructed vary, but are known to the art (Hoover & Hanna,1991, Immunotherapy by Active Specific Immunization: ClinicalApplications. In: Biologic Therapy of Cancer, (DeVita V. T., Hellman S.,Rosenberg, S. A., eds.) J. B. Lippincott Co., Philadelphia, pp. 670-682;McCabe et al., 1995, Minimal Determinant Expressed by a RecombinantVaccinia Virus Elicits Therapeutic Antitumor Cytolytic T LymphocyteResponses. Cancer Res. 55:1741-1747; Bauer et al., 1995, Identificationof H-2 Kb Binding and Immunogenic Peptides from Human Papilloma VirusTumour Antigens E6 and E7. Scand. J. Immunol. 42:317-323; Bocchia etal., 1995, Specific Binding of Leukemia Oncogene Fusion Protein Peptidesto HLA Class I Molecules. Blood 85:2680-2684; Peoples et al., 1995,Breast and Ovarian Cancer-Specific Cytotoxic T Lymphocytes Recognize theSame HER-2/Neu Derived Peptide. Proc. Natl. Acad. Sci. USA: 92:432-436;Yanuck et al., 1993, A Mutant P53 Tumor Suppressor Protein is a Targetfor Peptide-Induced CD8 Cytotoxic T-Cells. Cancer Res. 53:3257-3261),and are referenced herein in their entirety.

Of particular value, the invention allows the development andapplication of these tumor-specific therapies even when onlypremalignant tumors, early cancers, or occult cancers are present. Thus,the invention allows therapeutic intervention when tumor burden is low,immunologic function is relatively intact, and the patient is notcompromised, all increasing the potential for cure.

HYPOTHETICAL EXAMPLES OF THE INVENTION

In the following examples, illustrative hypothetical clinical cases arepresented to demonstrate the potential clinical use of the invention.

Case 1

A 26 year old asymptomatic hypothetical man presents for evaluationafter learning his 37 year old brother was recently diagnosed with coloncancer. Peripheral blood is drawn in order to use the invention toevaluate for the presence of extracellular CEA mRNA in the patient'splasma. Plasma extracellular RNA is extracted during invention Part A bythe Acid Guanidinium thiocyanate-Phenol-chloroform extraction method aspreviously described, followed by qualitative RT-PCR amplification ininvention Part B using CEA mRNA primers as previously described. Theamplification assay as previously described (Gerhard et al., 1994,Specific detection of carcinoembryonic antigen-expressing tumor cells inbone marrow aspirates by polymerase chain reaction. J. Clin. Oncol.12:725-729.) is performed in invention Part B. The final amplifiedproduct is detected by gel electrophoresis on a 3% agarose gel ininvention Part C. Results are positive in this patient indicating thepresence of CEA mRNA in the blood plasma.

CEA has been associated with colon cancer. While colon cancer is highlycurable if diagnosed at an early stage, it is fatal when diagnosed atadvanced metastatic stages. The positive results of the invention forthis patient, in the setting of a strongly positive family history forcolon cancer, are suggestive of either premalignant or malignant coloncancer. It is recommended that the patient undergo colonoscopy, and ifno lesion is found, receive surveillance more frequently than wouldnormally be given.

This hypothetical case illustrates how the invention can be used toscreen high risk patients for cancer, detect either premalignant ormalignant conditions prior to the metastatic state, and play a role inclinical management. While CEA mRNA is associated with other cancers,such as liver cancer, the addition of a multiplex panel approach usingthe invention to detect multiple different tumor-associatedextracellular RNA, including for example K-ras, P53, DCC, and APC RNA,enables clarification as to whether the CEA mRNA is likely associatedwith a colon tumor, and further, whether the findings are consistentwith a premalignant or a malignant tumor.

Case 2

A 33 year old hypothetical woman sees her local dermatologist afternoting a “bleeding mole” on her back. Local excision diagnoses amalignant melanoma of 0.3 millimeter depth. Wide surgical re-excision isperformed, and the patient is told she is likely cured and no furthertherapy is needed. Three months following her surgery the patient seeksa second opinion regarding the need for further therapy. Peripheralblood is drawn to evaluate her plasma for the presence of extracellulartyrosinase messenger RNA by the invention. Plasma extracellular RNA isextracted in invention Part A using the preferred TRI-Reagent method aspreviously described, followed by RT-PCR using nested primers fortyrosinase cDNA in invention Part B as previously described, with ELISAdetection in invention Part C. Invention results detect the presence oftyrosinase mRNA in the patient's plasma. Tyrosinase is common to bothnormal melanocytes and malignant melanoma. However, tyrosinase mRNA doesnot normally circulate in blood, and its presence in plasma indicateslatent malignant melanoma. Consequently, the patient is started onadjuvant therapy with interferon-alpha. Plasma extracellular tyrosinaseRNA levels are subsequently serially followed in a quantitative fashionusing the invention. Blood is drawn from the patient every two months,and plasma extracellular RNA is extracted in invention Part A using thesilica extraction method as previously described. Quantitative RT-PCRamplification for tyrosinase mRNA is then performed in invention Part Busing biotinylated primer using electrochemiluminescence based detectionin invention Part C. Invention data demonstrates a serial rise in thepatient's plasma extracellular tyrosinase mRNA levels. Consequent tothis data, the interferon is stopped, and the patient is enrolled intoan experimental adjuvant therapy protocol.

This hypothetical case illustrates several uses of the invention,including the detection of latent cancer, predicting prognosis andcancer recurrence following surgical excision, determining the need foradditional therapy, evaluating the benefit of therapy and the need tochange therapies, and evaluating prognosis of patients on therapy.

Case 3

A 76 year old hypothetical man is noted to have a pancreatic mass on CTscan imaging. His chest x-ray and colonoscopy are normal. The patientrefuses to consider surgery because of the significant surgical risks.He elects to receive patient-specific therapy made possible by use ofthe invention. Since X-ras mutations are present in 80-90% of pancreaticcancers, peripheral blood is drawn to evaluate for and characterizeextracellular mutant K-ras RNA circulating in plasma using theinvention. Plasma extracellular RNA is extracted in invention Part Ausing the TRI reagent extraction method as previously described,followed by RT-PCR in invention Part B, with high performance liquidchromatography detection in Part C. Mutant K-ras amplification productsare then separated following chromatography and the K-ras mutation issequenced using standard techniques as previously described. Detectionof mutant K-ras mRNA in the plasma confirms the likelihood of thepancreatic mass being a pancreatic cancer. Based upon the mutationsequence, a patient-specific therapy (i.e., specific to the patient'sown cancer) is developed, in this case a ras vaccine specific to themutant oncogene in this patient's pancreatic cancer. Alternatively,mutant K-ras specific protein, generated as previously described, may beused to develop a tumor-specific monoclonal antibody.

In this hypothetical case, the invention is used not only to helpconfirm a suspected diagnosis of pancreatic cancer, but to develop apatient-specific therapy. Patient-specific therapies—i.e., therapiesspecifically designed for a given patient's cancer, or a given type ofcancer, are possible when specific characteristics of the tumor arerecognized. Since the invention results in amplification of pure tumorproduct, it becomes possible to characterize the tumor, in this caseusing sequence analysis and/or transcription and translation. Thetechnological leap that the invention enables is that it allows tumorsto be characterized without the need for biopsy or surgery. Thus, itbecomes possible to treat tumors even before they become clinicallyevident, i.e., treating at latent stages, pre-recurrence stages, or evenpre-malignant stages. Early treatment of cancer before metastatic cellsenter the bloodstream increases the likelihood of cure.

Case 4

A 36 year old hypothetical woman who has three small children at homewas diagnosed with breast cancer two years ago. She had been treatedwith surgery followed by six months of chemotherapy. In addition, herblood serum has been serially evaluated for extracellular keratin 19mRNA using the invention in which serum extracellular kerain 19 mRNA isextracted in invention Part A using the silica extraction method,followed by RT-PCR amplification in invention Part B with ELISAdetection in invention Part C. Keratin 19 mRNA encodes for anintermediate filament protein not normally found in blood which canserve as a marker for breast cancer. While previous results for thispatient had been negative, her blood serum is now testing positive forextracellular keratin 19 mRNA by the invention, suggesting an impendingcancer recurrence. A multiplex panel for serum extracellular myc, ras,P53, EGFr, and Her-2-neu RNA is performed using the invention. This dataconfirms that tumor characteristics are identical to those of theoriginal breast cancer primary, confirming a recurrence rather than anew primary. Consequently, serum extracellular keratin 19 mRNA ismeasured in a quantitative fashion using a branched DNA signalamplification assay in invention Part B, with measurements performed 2months and 4 months later. Quantitative measurements indicate increasinglevels of keratin 19 mRNA, and allow extrapolation to predict thatclinical recurrence will be noted in approximately 2 years. Thisinformation allows both the physician and the patient to plan futuretherapeutic options in the context of the patient's current social andfamily situation.

This hypothetical case illustrates the use of the invention to monitorpatients following therapy for recurrence of their cancer, to determinecharacteristics of their tumor, and to predict prognosis. Breast cancerpatients have a high incidence of second primaries, but the inventionpermits delineation of primary versus recurrent cancer by using amultiplex panel approach to evaluate tumor characteristics. Furthermore,since quantitative analysis in invention Part B allows clarification ofprognosis, the patient is in a better position to plan therapy withinthe context of her social/family situation. Lastly, since the inventionallows detection of tumor-derived extracellular RNA, and does not dependupon the presence of circulating cancer cells, recurrence can bedetected at a very early stage (in this hypothetical case—2 years beforeclinical detection), which increases the likelihood of effectivetherapy.

1. A method of producing cDNA from extracellular mammalian total RNAextracted from a plasma or serum of a human or animal without cancer,wherein the method comprises the step of incubating said mammalian totalRNA with a reverse transcriptase.
 2. The method of claim 1, wherein thereverse transcriptase is a thermostable reverse transcriptase.
 3. Themethod of claim 1, wherein the mammalian total RNA is incubated with anoligodeoxynucleotide primer.
 4. The method of claim 3, wherein theoligodeoxynucleotide primer is a gene-specific primer.
 5. The method ofclaim 3, wherein the oligodeoxynucleotide primer is anoligodeoxythymidylate.
 6. The method of claim 3 or 4, wherein theoligodeoxynucleotide primer is detectably labeled.
 7. The method ofclaim 1, wherein a portion of the mammalian total RNA comprises at leasttwo or more RNA.
 8. The method of claim 7, wherein at least one or moreRNA are associated with a disease or condition.
 9. A method according toclaim 8, wherein the disease or condition is a premalignant disease orcondition.
 10. A method according to claim 1, wherein the human oranimal is at risk for a disease or condition.
 11. A method according toclaim 1, wherein the human or animal has a premalignant disease orcondition.
 12. The method of claim 1, wherein cDNA is further: a)hybridized; b) amplified in a qualitative or quantitative manner; c)sequenced; d) cloned; e) transcribed; f) used in a recombinant geneticconstruct; or g) otherwise manipulated.